Nuclear factor-κB (NF-κB) is a ubiquitous transcription factor that governs the expression of genes encoding cytokines, chemokines, growth factors, cell adhesion molecules and some acute phase proteins associated with various disease states. NF-κB is involved in immune and inflammatory reactions and its regulation contributes to a number of immunologically mediated diseases such as graft rejection, and diseases linked to inflammatory events such as autoimmune arthritis, asthma, septic shock, lung fibrosis, glomerulonephritis, artherosclerosis and AIDS.
DCs play a critical role in the initiation and regulation of immune responses and are instrumental in the induction and maintenance of tolerance (Banchereau and Steinman, Nature 392:245-252 (1998); Thomson and Lu, Transplantation 68:1-8 (1999)). DC activation is necessary for the role of DCs in the immune response and can be defined by two distinct processes, (1) maturation which involves the upregulation of MHC and costimulatory molecules, and (2) survival which involves the rescue of DCs from immediate apoptosis after the withdrawal of growth factors. See Rescigno et al., J. Exp. Med. 188:2175-2180 (1998). The mature DC expresses high levels of MHC class II and costimulatory molecules. In contrast, DCs with tolerogenic properties express low levels of costimulatory molecules and induce antigen-specific specific hyporesponsiveness by triggering T cell apoptosis. See Lu et al., Transplantation 60:1539-1545 (1995).
The inhibition of NF-κB can inhibit DC activation by blocking the maturation of DCs, a necessary process in DC activation. NF-κB is activated by a number of incoming signals from the cell surface. Once activated, NF-κB translocates into the nucleus and binds to the κB motif of the target gene. Nuclear translocation of NF-κB is associated with the expression of costimulatory molecules (e.g. CD40, CD86 and CD80) at the DC cell surface which correlates with the capacity of DCs to induce (or suppress) immune responses.
While DCs classically promote immune responses, they can be manipulated to induce antigen-specific hyporesponsiveness in vitro. The ability to manipulate the state of DC maturation in vitro has led to attempts to induce tolerance by administration of costimulatory molecule-deficient DCs in animal models of pancreatic islet cells or organ transplantation. See Fu et al., Transplantation 62:659-665 (1996); Rastellini et al., Transplantation 60:1366-1370 (1995); Lu et al., Transplantation 27:1808-1815 (1997); Gao et al., Immunology 98:159-170 (1999); Hirano et al., Transplant Proc. 32:260-264 (2000); Thomson and Lu, Transplantation 68:1-8 (1999). While these methods have had modest success, tolerance has not been achieved. This may be due to the late maturation/activation of DCs with upregulation of costimulatory molecules upon encountering a host microenvironment rich in pro-inflammatory mediators. The ability to manipulate the state of DC maturation may also be useful for the treatment of other diseases involving inflammatory events, such as autoimmune arthritis, asthma, septic shock, lung fibrosis, glomerulonephritis, artherosclerosis and AIDS.
U.S. Pat. No. 5,871,728 of Thomson et al., discloses a method for enhancing tolerogenicity to a foreign graft in a host mammal comprising propagating immature DCs from a mammalian source, culturing the immature DCs in the presence of a cytokine and administering the propagated immature DCs to the host. However, DCs cultured according to the method of U.S. Pat. No. 5,871,728, in which immature DCs are cultured in the presence of a cytokine alone, are likely to mature after encountering a host microenvironment. In fact, U.S. Pat. No. 5,871,728 also discloses a method for culturing mature immunostimulatory DCs which differs from the method of culturing immature DCs only by the addition of an extracellular matrix protein together with the cytokine. The inventors of the U.S. Pat. No. 5,871,728 patent acknowledge that the addition of the extracellular matrix protein during culturing creates an environment similar to that of the microenvironment in the host and thus leads to the maturation of the DCs. Therefore, it is very likely that the immature DCs of U.S. Pat. No. 5,871,728 will mature when introduced into a host cell microenvironment.
Genetic engineering of DCs to express immunosuppressive molecules has also been considered an attractive approach to alleviating of foreign graft rejection and autoimmune disorders. See Lu et al., J. Leukoc. Biol. 66:293-296 (1999). Adenoviral delivery of cytotoxic T lymphocyte antigen 4-immunoglobulin (CTLA4Ig) into DCs has been shown to promote DCs in vitro tolerogenicity and survival in allogeneic recipients. Lu et al., Gene Ther. 6:554-563 (1999). In addition, delivery of transforming growth factor-β (TGF-β) using an adenoviral vector prevents the reduction of DCs generally seen with adenovirus infection and also increases the numbers and prolongs the survival of the infected DCs in the spleen of a host to whom the DCs are administered. Lee et al., Transplantation 66:1810-1817 (1998).
Efforts have been made to develop genetic immunization with DCs infected with a viral vector expressing a gene of interest. Tuting et al. (J. Gene Med. 1:400-406 (1999)) have shown that DCs infected with a recombinant adenovirus encoding tyrosinase-related protein-2 (TRP2) induces anti-melanoma immunity. In addition, DCs have also been used for developing vaccinations against Epstein bar virus (EBV) by infecting DCs with an adenoviral vector encoding EBV antigens. Ranieri et al., J. Virol. 73:10416-10425 (1999).
While modification of DCs may be an attractive approach to the therapy of foreign graft rejection and autoimmune disorders, there are potential problems associated with such an approach. As noted above, tolerogenicity may be enhanced in a host by the administration of immature DCs which are hyporesponsive. However, infection of DCs with an adenoviral vector alone stimulates maturation of DCs and enhances the immunostimulatory capacity of DCs. See Rea et al., J. Virol. 73:10245-10253 (1999). In addition, it has been shown that infection of DCs with an adenovirus expressing eGFP enhanced costimulatory molecule expression and induction of CTL responses of both TGF-β and IL-4 in a dose dependent manner. See Lu et al., J. Leukocyte Bio. Supplement 2, abstract # B52 (1998).
Therefore, there is a need for a method for producing tolerogenic DCs which do not readily mature when introduced into a host. In addition, there is a need for a method of enhancing tolerogenicity in a host (such as autoimmune disease) using tolerogenic DCs wherein the tolerogenic DCs do not readily mature when introduced in the host microenvironment. Furthermore, there is a need for a method of producing tolerogenic DCs comprising a viral vector wherein said DCs maintain their tolerogenicity in the presence of the viral vector.